One of the mechanisms by which tumor cells metastasize is to secrete proteinases to degrade extracellular matrix (ECM) components between cells, so that they can break loose from the parent tumor and penetrate through the connective tissue barrier. Numerous correlative studies have demonstrated an increased level of proteolytic activity associated with malignant tumor in vivo and cell transformation in vitro. Our working hypothesis on tumor cell invasion has been that localized cell-surface proteinases initiate the cleavage of ECM proteins at cell-substrate contact sites, followed by a cascade of proteolytic activities, that allows cells to invade into the matrix. In order to understand the biochemical and cellular mechanisms involved in the invasive process of tumor cells, we propose to study the regulation of matrix degradation in a well-defined culture system of chicken embryonic fibroblasts (CEF) transformed by Rous sarcoma virus (RSV). In the RSV-transformed CEF culture system, in addition to plasminogen activator, two membrane-associated and transformation-induced proteinases have been identified at cellsubstrate contact sites. More recently, we have characterized a 7OkDa gelatinase secreted by RSV-transformed cells and shown its expression correlated to cell transformation. In this proposal, we will examine the expression and functional role of the 7OkDa gelatinase in transformed cells in vitro and in Rous sarcoma tumors in vivo using our highly specific, affinity purified, anti-catalytic antibodies against the avian 7OkD gelatinase. We will also investigate the biochemical characterization and biological function of the endogenous activator and inhibitor of the 7OkDa gelatinase. The cellular balance of the zymogen and active forms of the gelatinase, the activator, and the inhibitor will be evaluatcd and related directly to transformed cell behavior through the utilization of specific antibodies, enzyme activity assays, and the selective employment of the temperature-sensitive mutant of RSV. In addition, the fibronectin-degrading proteinase will be isolated and characterized. Hybridoma technology will be applied to gencrate monoclonal antibodies directed to the 45kDa proteinase. We will then use these immunoprobes to elucidate the possibly coordinate expression and catalytic activities of the 7OkDa gelatinase and 45kDa fibronectin-degrading proteinase in terms of hydrolyzation of various extracellular matrix proteins and effects on the invasive behavior of transformed cells.